Angiotensin converting enzyme (peptidyldipeptide hydrolase, hereinafter referred to as ACE) occupies a central role in the physiology of hypertension. The enzyme is capable of converting the decapeptide angiotensin I, having the sequence EQU AspArgValTyrIleHisProPheHisLeu
to an octapeptide, angiotensin II by removal of the carboxyterminal HisLeu. The symbols for various chemical entities are explained in the following table:
Ala=L-alanine PA1 Arg=L-arginine PA1 Asp=L-aspartic acid PA1 &lt;Glu=pyro-L-glutamic acid PA1 Gly=glycine PA1 Hip=Hippuric acid (Benzoyl glycine) PA1 His=L-histidine PA1 Ile=L-isoleucine PA1 Leu=L-leucine PA1 Phe=L-phenylalanine PA1 Pro=L-proline PA1 .DELTA.Pro=L-3,4-dehydroproline PA1 Ser=L-serine PA1 Trp=L-tryptophan PA1 Tyr=L-tyrosine PA1 Val=L-valine PA1 ACE=Angiotensin converting enzyme PA1 Hepes=N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid
Angiotensin I is formed by the action of the enzyme renin, an endopeptidase found in kidney, other tissues and plasma, acting on a serum .alpha.-2 globulin.
Blood pressure is affected by certain peptides found in the blood. One of these, angiotensin II, is a powerful pressor (blood pressure elevating) agent. Another, bradykinin, a nonapeptide with the sequence ArgProProGlyPheSerProPheArg is a powerful depressor (blood pressure lowering) agent. In addition to a direct pressor effect, angiotensin II stimulates release of aldosterone which tends to elevate blood pressure by causing retention of extracellular salt and fluids. Angiotensin II is found in measurable amount in the blood of normal humans. However, it is found at elevated concentrations in the blood of patients with renal hypertension.
The level of ACE activity is ordinarily in excess, in both normal and hypertensive humans, of the amount needed to maintain observed levels of angiotensin II. However, it has been found that significant blood pressure lowering is achieved in hypertensive patients by treatment with ACE inhibitors. [Gavras, I., and Vukovich, R. A., New Engl. J. Med. 291, 817 (1974)].
ACE is a peptidyldipeptide hydrolase. It catalyzes the hydrolysis of the penultimate peptide bond at the C-terminal end of a variety of acylated tripeptides and larger polypeptides having an unblocked .alpha.-carboxyl group. The action of ACE results in hydrolytic cleavage of the penultimate peptide bond from the carboxyl-terminal end yielding as reaction products a dipeptide and a remnant.
The reactivity of the enzyme varies markedly depending on the substrate. At least one type of peptide bond, having the nitrogen supplied by proline, is not hydrolyzed at all. The apparent Michaelis constant (Km) varies from substrate to substrate over several orders of magnitude. For general discussion of the kinetic parameters of enzyme catalyzed reactions, see Lehninger, A., Biochemistry, Worth Publishers, Inc., New York, 1970, pp. 153-157. Many peptides which are called inhibitors of the enzymatic conversion of angiotensin I to angiotensin II are in fact substrates having a lower Km than angiotensin I. Such peptides are more properly termed competitive substrates. Bradykinin is an example of a competitive substrate. In addition, a series of peptides isolated from the venom of Bothrops jararaca, a South American pit viper have been isolated, see Ferreira, S. H., et al., Biochemistry 9, 2583 (1970), which are strong competitive substrates of ACE.
The role of ACE in the pathogenesis of hypertension has stimulated interest in the venom peptides as possible antihypertensive drugs. The most potent is designated BPP.sub.9a (for Bradykinin Potentiating Peptide), and also termed SQ 20,881 by Ondetti, M. A., et al., Biochemistry 10, 4033 (1971), who have determined its sequence and have synthesized the peptide. The amino acid sequence of BPP.sub.9a is &lt;Glu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro. By convention, amino acid sequences are written and numbered left to right, from the N-terminal residue to the C-terminal residue. Substitutions are designated by position number. For example, .DELTA.Pro.sup.3 -BPP.sub.9a has the sequence of BPP.sub.9a except the L-3,4-dehydroproline is substituted for the proline at position three. Quantitatively, the inhibitory potency is expressed as the I.sub.50 value, defined as the concentration of inhibitor required to produce 50% inhibition of the enzyme in a standard assay system containing an approximately Km level of substrate. The I.sub.50 value for BPP.sub.9a is approximately 28 nM. For comparison, I.sub.50 for bradykinin is approximately 500 nM and I.sub. 50 for D-2-methyl-3-mercaptopropanoyl-L-proline (SQ 14,225, "captopril"), an orally effective antihypertensive compound, is approximately 23 nM.
Gavras, I., et al., New Engl. J. Med. 291, 817 (1974) and Clin. Sci. Mol. Med. 48, 575 (1975), have shown that BPP.sub.9a, injected intravenously at 1-4 mg/kg body weight, acts as a potent antihypertensive agent in patients with renin-related hypertension. The drug is also effective in lowering blood pressure of patients with essential hypertension when BPP.sub.9a administration follows treatment with moderate doses of diuretics. It is still not certain whether the blood pressure lowering effect of BPP.sub.9a is due to the prevention of the conversion of angiotensin I to the potent vasoconstrictor angiotensin II or to the prevention of the destruction of bradykinin by ACE.
In addition, many patients with essential hypertension respond favorably to treatment with BPP.sub.9a, see Case, D. B., et al., Am. J. Med. 61, 790 (1976) and Case, D. B., et al., New Engl. J. Med. 296, 641 (1977).
A major research and clinical testing effort has been focussed on the inhibitor SQ 14,225, since it is effective orally. However, in preliminary clinical trials, side reactions have occurred in a number of patients, although their cause is not known. The type of side reactions observed with SQ 14,225 have not been encountered previously with the clinical use of BPP.sub.9a. In view of the adverse reactions to SQ 14,225 experienced by some patients, and also because of instability of the drug in solution due to disulfide dimer formation, reversion to the clinical use of BPP.sub.9a as an antihypertensive agent may be warranted. Reversion to the clinical use of BPP.sub.9a will be particularly important for as many as 11% of the patients known to benefit from inhibitors of ACE, but who also respond adversely to SQ 14,225.
At present, little is known about what happens to BPP.sub.9a in vivo. A research program has been initiated to gain a better understanding of the metabolic fate and modes of elimination of BPP.sub.9a. In order to provide tritium-labeled BPP.sub.9a, novel analogs were synthesized that would be catalytically hydrogenated with tritium gas to yield [.sup.3 H]-BPP.sub.9a. Unexpectedly, the analogs proved to be highly potent ACE inhibitors in their own right, some of them being up to 20 times more potent than BPP.sub.9a itself.
A wide variety of peptide analogs of BPP.sub.9a has been synthesized. See Ondetti, et al., U.S. Pat. No. 3,832,337, issued Aug. 27, 1974; Cushman, D. W., et al., Experientia 29, 1032 (1973) and Pluscec, I., et al., Peptides 1972, Proc. 12th Europ. Peptide Symp. (H. Hanson and H. D. Jakubke, eds.) North Holland Publ. Co., Amsterdam, 1972, p. 403. From these studies it appears that analogs of similar inhibitory potency can be provided by substituting a cyclopentylcarbonyl for the pyroglutamyl residue, phenylalanine for the tryptophan residue, glycine or histidine for the arginine residue and phenylalanine for the isoleucine residue.
Other substitutions, single and in combination, have been investigated, as disclosed in U.S. Pat. No. 3,832,337. Modest increases in potency have been observed, and a partial pattern of permissible substitutions has been developed. However, few substitutions of proline analogs for any of the proline residues have been tested. In one instance, substitution of a pyrrolidinyl reside at Pro.sup.9 resulted in a substantial loss of inhibitory potency. Substitution of an alanine residue at Pro.sup.8 somewhat improved inhibitory potency, by a factor of 2.75. (See Cushman, D. W., et al., supra). However, these workers concluded generally that at least the C-terminal pentapeptide sequence -Pro-Gln-Ile-Pro-Pro is required for significant inhibition.
Derivatives of bradykinin having a proline in the 2, 3 or 7 position substituted by L-3,4-dehydroproline have been studied for physiological effect on the contractile response of rat uterus and guinea pig ileum. See Fisher, G. H., et al., Arch. Biochem. Biophys. 189, 81 (1978). The analogs .DELTA.pro.sup.2 -bradykinin and .DELTA.pro.sup.3 -bradykinin were approximately as effective as bradykinin, while .DELTA.pro.sup.7 -bradykinin was only about 25% as effective.